Treatment for methamphetamine cardiovascular disease

ABSTRACT

A method of treating or preventing methamphetamine related endothelial dysfunction in a patient comprising administering to the patient an effective dose of a pharmacologic composition; the composition comprising a therapeutic, the therapeutic including a hydrogen sulfide (H 2 S) donor, or a salt, solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug or analogs thereof. The H 2 S donor may be one of sodium sulfide, diallyl trisulfide, diallyl disulfide, acillin, sugammadex, sulfanilamide, disulfram, sulfonamide, sulfinates, sulfoxides, persulfides, polysulfides, and sulfones. The H 2 S donor may be sugammadex. The H 2 S donor may be administered in a dosage of between 0.5 mg/kg and 10.0 mg/kg.

CROSS REFERENCE TO RELATED APPLICATIONS/PRIORITY

This application is a continuation of U.S. Ser. No. 17/397,973 filedAug. 9, 2021, which claims the benefit of U.S. Provisional PatentApplication No. 63/063,225 filed Aug. 8, 2020, all of which are herebyincorporated by reference in their entirety.

BACKGROUND

Methamphetamine was first synthesized in 1893 by the Japanese chemistNagai Nagayoshi. Methamphetamine is a more potent version ofamphetamine, first synthesized in Germany in 1887. During World War II,the Axis and Allied forces used methamphetamine and amphetamine toextend wakefulness, and in the postwar period both agents were used asdiet pills, before their destructive and addictive nature were fullyunderstood. Methamphetamine is available in various forms includingliquid, powder, and as a crystalline substance, first synthesized inJapan by Akira Ogata in 1919, which can be smoked. Both when injectedintravenously as well as when smoked, high levels of the drug arerapidly achieved in the circulation. The main effects areneurocognitive—both a euphoric and energized state as well as psychosis,depression, and other neuropsychiatric and cognitive sequelae(dizziness, anxiety, apathy, depression, aggression, cognitiveimpairments, personality changes, mania, psychosis). Methamphetamineabusers typically go on “meth binges” lasting days.

Methamphetamine (methamphetamine) is a markedly addictive illicit drugand has severe psychological, and social risks that affect differentethnic groups worldwide. Statistics from the National Survey on Drug Useand Health (NSDUH) reveal that an estimated 24.6 million Americans ages12 or older have used methamphetamine in their lifetimes for non-medicalreasons. There are significant social and health hazards associated withmethamphetamine usage. Recent studies have revealed increased prevalenceof cardiovascular disease (CVD) including hypertension, vasospasm,cardiomegaly, arrhythmias, left ventricular hypertrophy, myocardialinfarction, and coronary artery disease at a young age. Methamphetaminehas effects on multiple organ systems including cardiovascularcomplications that are the second leading cause of death due tomethamphetamine use. Importantly, methamphetamine use disproportionallyincreases cardiovascular-related morbidity and mortality in up tothree-fourths of its users. Several clinical and postmortem studiesassociate the use of methamphetamine with cardiovascular disease leadingto death. However, the understanding of methamphetamine-relatedcardiovascular implications and the underlying molecular mechanismsremain poorly understood, which could explain why some traditionalcardiac medications prescribed to methamphetamine users to treatcardiovascular disease have been ineffectual.

SUMMARY

Wherefore, it is an object of the present invention to overcome theabove-mentioned shortcomings and drawbacks associated with the currenttechnology.

The presently disclosed invention is related to therapeutics and methodsof treating or preventing methamphetamine related endothelialdysfunction in a patient comprising administering to the patient aneffective dose of a pharmacologic composition the composition comprisinga therapeutic the therapeutic including a hydrogen sulfide (H₂S) donor.According to a further embodiment the H₂S donor is one of sodiumsulfide, diallyl trisulfide, diallyl disulfide, allicin, sugammadex,sulfanilamide, disulfiram, sulfonamide, sulfinates, sulfoxides,persulfides, polysulfides, and sulfones. According to a furtherembodiment the H₂S donor is sugammadex. According to a furtherembodiment the H₂S donor is administered in a dosage of between 0.5mg/kg and 10.0 mg/kg. According to a further embodiment the H₂S donor isadministered exactly once in a dosage period, the dosage period beingbetween 1 day and 30 days. According to a further embodiment the dosageperiod is seven days. According to a further embodiment the therapeuticis administered in one of oral, intravenous, and transdermal pathways.According to a further embodiment the therapeutic is administeredorally. According to a further embodiment the therapeutic has an entericcoating. According to a further embodiment the therapeutic isadministered intravenously. According to a further embodiment thepatient has one of atherosclerosis, hypertension, myocardial infarction,and diabetes, and cardiovascular disease, or a precondition thereof.According to a further embodiment, the composition further comprises oneof cystathionine gamma lyase or a salt, solvate, ester, amide,clathrate, stereoisomer, enantiomer, prodrug, or analogs thereof.

The presently disclosed invention further relates to therapeutics andmethods of treating an endothelial dysfunction related disease in amethamphetamine patient comprising administering to the patient aneffective dose of a pharmacologic composition, the compositioncomprising a therapeutic, and the therapeutic including a hydrogensulfide (H₂S) donor. According to a further embodiment, the relateddisease is one of atherosclerosis, hypertension, myocardial infarction,and diabetes, and cardiovascular disease, or a precondition thereof.According to a further embodiment, the method further comprisesadministering a second, non-H₂S donor therapeutic for the treatment ofthe related disease.

The presently disclosed invention further relates to methods oftreatment and pharmaceutical compositions comprising a therapeutic, thetherapeutic including a hydrogen sulfide (H₂S) donor formulated for oneof oral and peritoneal administration and being in a dosage of between1.0 mg and 100.0 mg of H₂S donor. According to a further embodiment, thecomposition includes an exact dosage of between 5.0 mg and 60 mg of theH₂S donor, and between 10 mg and 30 mg of the H₂S donor. According to afurther embodiment, composition further comprises cystathionine gammalyase or a salt, solvate, ester, amide, clathrate, stereoisomer,enantiomer, prodrug, or analogs thereof. According to a furtherembodiment, the composition includes an exact dosage of between 1.0 mgand 100 mg, 5.0 mg and 60 mg, and between 10 mg and 30 mg ofcystathionine gamma lyase or a salt, solvate, ester, amide, clathrate,stereoisomer, enantiomer, prodrug, or analogs thereof.

The present invention relates to pharmaceutical compositions of atherapeutic (e.g., a hydrogen sulfide (H₂S) donor), or apharmaceutically acceptable salt, solvate, ester, amide, clathrate,stereoisomer, enantiomer, prodrug or analogs thereof, and use of thesecompositions for the treatment of endothelial dysfunction and diseasesand conditions that are causes of endothelial dysfunctions, such ascardiovascular complications including atherosclerosis, hypertension,myocardial infarction, and diabetes, and cardiovascular disease.Examples of H₂S donors include sodium sulfide, diallyl trisulfide,diallyl disulfide, allicin, sugammadex, sulfanilamide, disulfiram,sulfonamide, sulfinates, sulfoxides, persulfides, polysulfides, andsulfones.

In some embodiments, the therapeutic, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, is administered as a pharmaceuticalcomposition that further includes a pharmaceutically acceptableexcipient.

In some embodiments, administration of the pharmaceutical composition toa human results in a peak plasma concentration of the therapeuticbetween 0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

In some embodiments, the peak plasma concentration of the therapeutic ismaintained for up to 14 hours. In other embodiments, the peak plasmaconcentration of the therapeutic is maintained for up to 1 hour.

In some embodiments, the condition is an endothelial dysfunction.

In certain embodiments, the endothelial dysfunction is mild to moderateendothelial dysfunction.

In further embodiments, the endothelial dysfunction is moderate tosevere endothelial dysfunction.

In other embodiments, the therapeutic is administered at a dose that isbetween 0.05 mg-5 mg/kg weight of the human.

In certain embodiments, the pharmaceutical composition is formulated fororal administration.

In other embodiments, the pharmaceutical composition is formulated forextended release.

In still other embodiments, the pharmaceutical composition is formulatedfor immediate release.

In some embodiments, the pharmaceutical composition is administeredconcurrently with one or more additional therapeutic agents for thetreatment or prevention of the endothelial dysfunction.

In some embodiments, the therapeutic, or a pharmaceutically acceptablesalt, solvate, or prodrug thereof, is administered as a pharmaceuticalcomposition that further includes a pharmaceutically acceptableexcipient.

In some embodiments, administration of the pharmaceutical composition toa human result in a peak plasma concentration of the therapeutic between0.05 μM-10 μM (e.g., between 0.05 μM-5 μM).

In some embodiments, the peak plasma concentration of the therapeutic ismaintained for up to 14 hours. In other embodiments, the peak plasmaconcentration of the therapeutic is maintained for up to 1 hour.

In other embodiments, the therapeutic is administered at a dose that isbetween 0.05 mg-5 mg/kg weight of the human.

In certain embodiments, the pharmaceutical composition is formulated fororal administration.

In other embodiments, the pharmaceutical composition is formulated forextended release.

In still other embodiments, the pharmaceutical composition is formulatedfor immediate release.

As used herein, the term “delayed release” includes a pharmaceuticalpreparation, e.g., an orally administered formulation, which passesthrough the stomach substantially intact and dissolves in the smalland/or large intestine (e.g., the colon). In some embodiments, delayedrelease of the active agent (e.g., a therapeutic as described herein)results from the use of an enteric coating of an oral medication (e.g.,an oral dosage form).

The term an “effective amount” of an agent, as used herein, is thatamount sufficient to effect beneficial or desired results, such asclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied.

The terms “extended release” or “sustained release” interchangeablyinclude a drug formulation that provides for gradual release of a drugover an extended period of time, e.g., 6-12 hours or more, compared toan immediate release formulation of the same drug. Preferably, althoughnot necessarily, results in substantially constant blood levels of adrug over an extended time period that are within therapeutic levels andfall within a peak plasma concentration range that is between, forexample, 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1 μM.

As used herein, the terms “formulated for enteric release” and “entericformulation” include pharmaceutical compositions, e.g., oral dosageforms, for oral administration able to provide protection fromdissolution in the high acid (low pH) environment of the stomach.Enteric formulations can be obtained by, for example, incorporating intothe pharmaceutical composition a polymer resistant to dissolution ingastric juices. In some embodiments, the polymers have an optimum pH fordissolution in the range of approx. 5.0 to 7.0 (“pH sensitivepolymers”). Exemplary polymers include methacrylate acid copolymers thatare known by the trade name Eudragit® (e.g., Eudragit® L100, Eudragit®S100, Eudragit® L-30D, Eudragit® FS 30D, and Eudragit® L100-55),cellulose acetate phthalate, cellulose acetate trimellitiate, polyvinylacetate phthalate (e.g., Coateric®), hydroxyethylcellulose phthalate,hydroxypropyl methylcellulose phthalate, or shellac, or an aqueousdispersion thereof. Aqueous dispersions of these polymers includedispersions of cellulose acetate phthalate (Aquateric®) or shellac(e.g., MarCoat 125 and 125N). An enteric formulation reduces thepercentage of the administered dose released into the stomach by atleast 50%, 60%, 70%, 80%, 90%, 95%, or even 98% in comparison to animmediate release formulation. Where such a polymer coats a tablet orcapsule, this coat is also referred to as an “enteric coating.”

The term “immediate release” includes where the agent (e.g.,therapeutic), as formulated in a unit dosage form, has a dissolutionrelease profile under in vitro conditions in which at least 55%, 65%,75%, 85%, or 95% of the agent is released within the first two hours ofadministration to, e.g., a human. Desirably, the agent formulated in aunit dosage has a dissolution release profile under in vitro conditionsin which at least 50%, 65%, 75%, 85%, 90%, or 95% of the agent isreleased within the first 30 minutes, 45 minutes, or 60 minutes ofadministration.

The term “pharmaceutical composition,” as used herein, includes acomposition containing a compound described herein (e.g., an H₂S donor,or any pharmaceutically acceptable salt, solvate, or prodrug thereof),formulated with a pharmaceutically acceptable excipient, and typicallymanufactured or sold with the approval of a governmental regulatoryagency as part of a therapeutic regimen for the treatment of disease ina mammal.

Pharmaceutical compositions can be formulated, for example, for oraladministration in unit dosage form (e.g., a tablet, capsule, caplet,gelcap, or syrup); for topical administration (e.g., as a cream, gel,lotion, or ointment); for intravenous administration (e.g., as a sterilesolution free of particulate emboli and in a solvent system suitable forintravenous use); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, includes anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,cross-linked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, maltose,mannitol, methionine, methylcellulose, methyl paraben, microcrystallinecellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone,pregelatinized starch, propyl paraben, retinyl palmitate, shellac,silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodiumstarch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, andxylitol.

The term “pharmaceutically acceptable prodrugs” as used herein, includesthose prodrugs of the compounds of the present invention which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and animals with undue toxicity, irritation,allergic response, and the like, commensurate with a reasonablebenefit/risk ratio, and effective for their intended use, as well as thezwitterionic forms, where possible, of the compounds of the invention.

The term “pharmaceutically acceptable salt,” as use herein, includesthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds of the invention orseparately by reacting the free base group with a suitable organic orinorganic acid. Representative acid addition salts include acetate,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemi sulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, oleate, oxalate, palmitate, pamoate,pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,toluenesulfonate, undecanoate, valerate salts, and the like.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like, as well asnontoxic ammonium, quaternary ammonium, and amine cations, including,but not limited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine,and the like.

The terms “pharmaceutically acceptable solvate” or “solvate,” as usedherein, includes a compound of the invention wherein molecules of asuitable solvent are incorporated in the crystal lattice. A suitablesolvent is physiologically tolerable at the administered dose. Forexample, solvates may be prepared by crystallization, recrystallization,or precipitation from a solution that includes organic solvents, water,or a mixture thereof. Examples of suitable solvents are ethanol, water(for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone(NMP), dimethyl sulfoxide (DMSO), N,N′-dimethylformamide (DMF),N,N′-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, and the like. When water is the solvent, the solvate isreferred to as a “hydrate.”

The term “prevent,” as used herein, includes prophylactic treatment ortreatment that prevents one or more symptoms or conditions of a disease,disorder, or conditions described herein (e.g., an endothelialdysfunction). Treatment can be initiated, for example, prior to(“pre-exposure prophylaxis”) or following (“post-exposure prophylaxis”)an event that precedes the onset of the disease, disorder, orconditions. Treatment that includes administration of a compound of theinvention, or a pharmaceutical composition thereof, can be acute,short-term, or chronic. The doses administered may be varied during thecourse of preventive treatment.

The term “prodrug,” as used herein, includes compounds which are rapidlytransformed in vivo to the parent compound of the above formula.Prodrugs also encompass bioequivalent compounds that, when administeredto a human, lead to the in vivo formation of therapeutic. A thoroughdiscussion is provided in T. Higuchi and V. Stella, Pro-drugs as NovelDelivery Systems, Vol. 14 of the A.C.S. Symposium Series, and Edward B.Roche, ed., Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987, each of which isincorporated herein by reference. Preferably, prodrugs of the compoundsof the present invention are pharmaceutically acceptable.

As used herein, and as well understood in the art, “treatment” includesan approach for obtaining beneficial or desired results, such asclinical results. Beneficial or desired results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions; diminishment of extent of disease, disorder, or condition;stabilized (i.e. not worsening) state of disease, disorder, orcondition; preventing spread of disease, disorder, or condition; delayor slowing the progress of the disease, disorder, or condition;amelioration or palliation of the disease, disorder, or condition; andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. As used herein, theterms “treating” and “treatment” can also include delaying the onset of,impeding, or reversing the progress of, or alleviating either thedisease or condition to which the term applies, or one or more symptomsof such disease or condition.

The term “unit dosage forms” includes physically discrete units suitableas unitary dosages for human subjects and other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with any suitablepharmaceutical excipient or excipients.

As used herein, the term “plasma concentration” includes the amount oftherapeutic present in the plasma of a treated subject (e.g., asmeasured in a rabbit using an assay described below or in a human).

The disclosed invention further relates to the identification ofhydrogen sulfide and CSE as a key target of methamphetamine dependentcardiovascular dysfunction.

The disclosed invention further relates to disclosure of molecularmechanisms involved in methamphetamine reduction of H₂S via inhibitionof CSE gene expression.

The disclosed invention further relates to disclosure thatmethamphetamine decreases H₂S levels, which then contributes to NOdysregulation.

The disclosed invention further relates to creating the therapeuticapproaches and verifying that both pharmacologic and genetic therapeuticapproaches attenuate methamphetamine endothelial dysfunction.

The disclosed invention further relates to experimentally showing theclinical utility of H₂S based therapeutic approaches for cardiovasculardysfunction in methamphetamine abusers.

The disclosed invention further relates to the discovery thatmethamphetamine increases pro-aging gene expression.

The disclosed invention further relates to the discovery that Sugammadexincreases plasma sulfide levels in wild type and CSE knockout mice totherapeutically significant levels at fractional doses.

The disclosed invention further relates to the discovery that Sugammadexincreases human plasma sulfide levels and CSE activity in humans totherapeutically significant levels at fractional doses.

Various objects, features, aspects, and advantages of the presentinvention will become more apparent from the following detaileddescription of preferred embodiments of the invention, along with theaccompanying drawings in which like numerals represent like components.The present invention may address one or more of the problems anddeficiencies of the current technology discussed above. However, it iscontemplated that the invention may prove useful in addressing otherproblems and deficiencies in a number of technical areas. Therefore, theclaimed invention should not necessarily be construed as limited toaddressing any of the particular problems or deficiencies discussedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The accompanying drawings, which are incorporated in and constitute apart of the specification, illustrate various embodiments of theinvention and together with the general description of the inventiongiven above and the detailed description of the drawings given below,serve to explain the principles of the invention. It is to beappreciated that while the accompanying drawings are to scale, theemphasis is instead placed on illustrating the principles of theinvention. The invention will now be described, by way of example, withreference to the accompanying drawings in which:

FIGS. 1A-1F show methamphetamine decreases plasma total sulfide and NOmetabolites in mice;

FIGS. 2A-2F show methamphetamine decreases murine CSE expression andeNOS phosphorylation;

FIGS. 3A-3C show methamphetamine decreases CSE Protein Expression inMouse muscle tissue;

FIGS. 4A-4B show methamphetamine decreases CSE enzyme activity in mousetissue;

FIGS. 5A-5F show methamphetamine increases oxidative stress andinflammation in mouse skeletal muscle;

FIGS. 6A-6D show methamphetamine causes endothelial cell dysfunction inmice;

FIGS. 7A and 7B show exogenous sulfide/CSE expression rescuesmethamphetamine-mediated vascular dysfunction in mice;

FIGS. 8A and 8B show exogenous sulfide/CSE rescues methamphetaminemediated cardiac dysfunction;

FIGS. 9A-9D show sulfide or CSE expression corrects methamphetaminedefects in tissue aging genes;

FIGS. 10A-10C show the effect of methamphetamine on CSE proteinexpression in humans;

FIGS. 11A and 11B show methamphetamine decreases CSE activity in humanplasma and endothelial cells;

FIG. 12 shows sulfide release with Sugammadex in WT and CSEKO mice; and

FIGS. 13A-3C show sulfide release and CSE activity with Sugammadex inHuman Plasma.

DETAILED DESCRIPTION

The present invention will be understood by reference to the followingdetailed description, which should be read in conjunction with theappended drawings. It is to be appreciated that the following detaileddescription of various embodiments is by way of example only and is notmeant to limit, in any way, the scope of the present invention. In thesummary above, in the following detailed description, in the claimsbelow, and in the accompanying drawings, reference is made to particularfeatures (including method steps) of the present invention. It is to beunderstood that the disclosure of the invention in this specificationincludes all possible combinations of such particular features, not justthose explicitly described. For example, where a particular feature isdisclosed in the context of a particular aspect or embodiment of theinvention or a particular claim, that feature can also be used, to theextent possible, in combination with and/or in the context of otherparticular aspects and embodiments of the invention, and in theinvention generally. The terms “comprise(s),” “include(s),” “having,”“has,” “can,” “contain(s),” and grammatical equivalents and variantsthereof, as used herein, are intended to be open-ended transitionalphrases, terms, or words that do not preclude the possibility ofadditional acts or structures. are used herein to mean that othercomponents, ingredients, steps, etc. are optionally present. Forexample, an article “comprising” (or “which comprises”) components A, B,and C can consist of (i.e., contain only) components A, B, and C, or cancontain not only components A, B, and C but also one or more othercomponents. The singular forms “a,” “and” and “the” include pluralreferences unless the context clearly dictates otherwise. Wherereference is made herein to a method comprising two or more definedsteps, the defined steps can be carried out in any order orsimultaneously (except where the context excludes that possibility), andthe method can include one or more other steps which are carried outbefore any of the defined steps, between two of the defined steps, orafter all the defined steps (except where the context excludes thatpossibility).

The term “at least” followed by a number is used herein to denote thestart of a range beginning with that number (which may be a range havingan upper limit or no upper limit, depending on the variable beingdefined). For example, “at least 1” means 1 or more than 1. The term “atmost” followed by a number is used herein to denote the end of a rangeending with that number (which may be a range having 1 or 0 as its lowerlimit, or a range having no lower limit, depending upon the variablebeing defined). For example, “at most 4” means 4 or less than 4, and “atmost 40%” means 40% or less than 40%. When, in this specification, arange is given as “(a first number) to (a second number)” or “(a firstnumber)-(a second number),” this means a range whose lower limit is thefirst number and whose upper limit is the second number. For example, 25to 100 mm means a range whose lower limit is 25 mm, and whose upperlimit is 100 mm.

The embodiments set forth the below represent the necessary informationto enable those skilled in the art to practice the invention andillustrate the best mode of practicing the invention. For themeasurements listed, embodiments including measurements plus or minusthe measurement times 5%, 10%, 20%, 50% and 75% are also contemplated.For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

In addition, the invention does not require that all the advantageousfeatures and all the advantages of any of the embodiments need to beincorporated into every embodiment of the invention.

Turning now to FIGS. 1A-14C, a brief description concerning the variouscomponents of the present invention will now be briefly discussed.

Vascular endothelium plays a central role in the maintenance ofcardiovascular homeostasis. Disruption of endothelial function,clinically referred to as endothelial dysfunction, is a keypathophysiological mediator of nearly all cardiovascular disease.Endothelial dysfunction is a hallmark prerequisite of cardiovascularcomplications including atherosclerosis, hypertension, myocardialinfarction, and diabetes. Methamphetamine can have adverse andpotentially fatal effects on arteries and blood vessels, which resultsin increased blood pressure, inflammation and cardiovascular dysfunctionincluding atherosclerosis. Methamphetamine induces pro-inflammatorysignaling responses and increases the production of reactive oxygenspecies that are detrimental to the cardiovascular system. Whilemethamphetamine may induce oxidative stress contributing tocardiovascular dysfunction, the specific mechanisms, reactive oxygenspecies involved, and the molecular trigger of events leading to thisresponse remained completely unknown in the current technology.

Hydrogen sulfide (H₂S) and nitric oxide (NO) are gaseous signalingmolecules that serve important roles in regulating endothelial andcardiovascular health. Cystathionine gamma lyase (CSE) and H₂S playcritical regulatory roles in various cardiovascular pathophysiologicalfunctions including vasorelaxation, protection from oxidative damage andcytoprotection. Vascular H₂S bioavailability directly influences NObioavailability. However, the underlying molecular mechanisms ofmethamphetamine mediated endovascular dysfunction remained elusive inthe current technology. Additionally, no information previously existedregarding the role of CSE/H₂S/NO alterations in clinical vasculardisease conditions of methamphetamine users. Also, prolonged exposure ofmethamphetamine can induce pro-oxidant and pro-inflammatory events andsignaling that trigger endothelial dysfunction, aggravating thecardiovascular complications. Importantly, previous studies aimed atunderstanding a relationship between methamphetamine and NO have beenfocused on neuronal injury with no clear understanding of impacts invascular function. This current disclosure describes the molecularmechanisms underlying methamphetamine-mediated H₂S/NO signaling,oxidative stress and inflammation leading to vascular dysfunction, andtherapeutics and methods of treatment based on such discoveries.

Materials and Methods:

2.1 Chemicals and Reagents: Chemicals and tissue culture reagents,including Methamphetamine hydrochloride (methamphetamine) were obtainedfrom Sigma unless otherwise noted. Anhydrous sodium sulfide waspurchased from Alfa-Aesar Inc. Anti-CD31 antibody was from BDBiosciences (San Jose, CA, USA), and anti-α-SMA antibody was obtainedfrom Sigma-Aldrich. VECTASHIELD PLUS DAPI was from Vector Laboratories.All secondary fluorophore-labeled antibodies were obtained from JacksonImmunoresearch Inc (West Grove, PA, USA). Human umbilical veinendothelial cells (HUVECs) were from Lifeline Cell Technology, CA, USA.

2.2 Cell culture and treatments: Human umbilical vein endothelial cells(HUVECs) were purchased from Lifeline Cell Technology (Cat. No. FC-0044)and cultured in VascuLife® Basal Medium (Cat. No. LM-0002) supplementedwith the appropriate LifeFactors® Kit (No. LL-0003). All cells weregrown in tissue culture flasks at 37° C. and 5% CO₂. HUVECs frompassages 2-4 were used in the experiments.

2.3 Mouse model and treatment routes: Twelve-week-old male WT (C57BL6/J)and CSE Tg male mice were used to study ‘Binge’ methamphetamineadministration effects on endovascular function. Mice were randomlyassigned to different experimental groups by one investigator and weretreated and evaluated by a second blinded investigator. NaHS drinkingsolutions were made by dissolving the appropriate amount of NaHS withthe appropriate volume of tap water for final NaHS concentration (30Sulfide donor, Na₂S (30 μM) or just water was administered in thedrinking water of mice treated with either methamphetamine or salineduring the length of the study. Mice were housed at the Louisiana StateUniversity Health Sciences Center-Shreveport animal resources facility,which is accredited by the Association for Assessment and Accreditationof Laboratory Animal Care International. All animal studies wereapproved by the LSU Institutional Animal Care and Use Committee (LSUIACUC Protocol #P-08-021) and in accordance with the Guide for the Careand Use of Laboratory Animals published by the National Institutes ofHealth.

2.4 ‘Binge and crash’ mouse methamphetamine model: Twelve-week-old maleWT (C57BL6/J) male mice were used for these experiments. Mice wereexposed to methamphetamine according to a known protocol that modelsbinge methamphetamine exposure in humans. C57BL/6 male mice received 0-6mg/kg methamphetamine (Methamphetamine HCl, Sigma-Aldrich, St. Louis,MO) through subcutaneous (s.c.) injection five days in a week for fourweeks. Methamphetamine was dissolved in a sterile saline (Sigma-Aldrich)solution (0.9% w/v NaCl). Vehicle treated mice received the same volumeof saline at all-time points for four weeks. Briefly,methamphetamine—exposed and saline—control mice were injectedsubcutaneously with methamphetamine or saline in a volume of 5 ml/kgwith an insulin syringe. Alternating sites of injection were used toavoid any possible damage to tissue and possible stress of the animal.The dose of methamphetamine was escalated over the course of the firstcycle, which occurs during the first week of injections (days 1-5)followed by three weeks of repeated cycles of meth injections (days8-12, 15-19, and 22-26). Mice received 4 injections per day, 2 hoursapart with doses of meth including 0, 1, 2, 3, 4, 5, and 6 mg/kgsubcutaneously. At the end of the 4 weeks, mice were sacrificed byisoflurane overdose, plasma and tissue were harvested.

2.5 Flow mediated dilation (FMD): Vascular function was determined bymouse FMD. Experimental cohorts of Control (saline), methamphetamine(0-6 mg/kg) with or without sulfide (30 μM), were treated for 4 weeks,and subjected to FMD. Briefly, mice were anesthetized with isofluraneand fur was removed from the hindlimbs. The animals were then placed ona warmed ultrasound table equipped with ECG. A vascular occluder (5 mmdiameter, Harvard Apparatus) was placed around the proximal hindlimb toinduce transient occlusion of the vessels of the distal hindlimb as anischemic trigger. The Doppler ultrasound probe (VEVO 3100, VisualSonics)was manually aligned over the femoral artery, distal to the occluder, totake baseline recordings of the blood vessel for diameter (M mode) andmean velocity (PW mode). The vascular occluder was inflated manuallywith an air-filled syringe for 1-minute, and deflated. Measurements ofdiameter and blood flow velocity were recorded for 180 s at 30 sintervals. The recorded loops were analyzed by Vevo LAB analysissoftware.

2.6 Human blood collection: Human subjects were interviewed, and medicalrecord data were collected for analysis of methamphetamine-use. Bloodsamples were collected from already-established catheterization into 6mL BD vacutainer tubes with lithium heparin. Samples were transported tothe lab within 15 min on ice and were centrifuged at 1500 RCF for 4 minat 4° C.

2.7 Measurement of biological pools of H₂S: Plasma samples from mouseand human subjects were analyzed for free sulfide, acid-labile sulfide(ALS), bound sulfane sulfur (BSS), and total sulfide levels using themonobromobimane (MBB) method as the inventors have previously reported.Free sulfide was measured using 50 μl of plasma with MBB; whereas fordetection of ALS and BSS, 50 μl of plasma was added separately into twosets of 4 mL BD vacutainer tubes. Four hundred fifty microliters of 100mM phosphate buffer (pH 2.6, 0.1 mM DTPA) was added to one tube [acidlabile reaction] and 450 μl of 100 mM phosphate buffer (pH 2.6, 0.1 mMDTPA) plus 1 mM TCEP was added to the second tube [total sulfidereaction]. Following a 30-min incubation on a nutator, the reactionliquid was removed, and the evolved sulfide gas subsequently trapped byadding 500 μl of 100 mM Tris-HCl buffer (pH 9.5, 0.1 mM DTPA) into theBD vacutainer tube and incubated again for 30 min on a nutator mixer.The trapping solutions were removed, and sulfide levels measured usingthe MBB method as the inventors have previously reported. Determinationof ALS was made by reacting plasma samples with acidic phosphate bufferalone and subsequent trapping of evolved sulfide. Measurement of BSS wasdetermined by subtracting the acid labile value from the total sulfideprotocol containing TCEP reductant treatment under acidic conditions.Total sulfide levels were directly obtained from the total sulfidereaction.

2.8 NO metabolite measurements: NO metabolites (NOx) were measured usingan ozone-based chemiluminescent assay (Sievers Nitric Oxide Analyzer280i, Weddington, NC) as described previously. Plasma and skeletalmuscle tissue samples were collected in NO preservation buffer (1.25mol/L potassium ferricyanide, 56.9 mmol/L N-ethylmaleimide, 6% NonidetP-40 substitute in PBS). Aliquots of samples were tested for freenitrite and sulfanilamide resistance following addition of an acidicsulfanilamide solution to a final concentration of 0.5% v/v and sittingin the dark for 15 min prior to injection into the analyzer.

2.9 Cystathionine γ-lyase (CSE) activity measurement: CSE activity wasmeasured as previously reported. Plasma or tissue lysates were incubatedwith 2 mM cystathionine, 0.25 mM pyridoxal 5′-phosphate in 100 mMTris-HCl buffer (pH 8.3) for 60 min at 37° C. 10% Trichloroacetic acidwas added into reaction mixture. After centrifugation, the supernatantwas mixed with 1% ninhydrin reagent and incubated for 5 min in aboiling-water bath. After heating, the solution was cooled on ice for 2minutes and color reaction development measured 20 minutes at 455 nmusing a spectrophotometer (Biotek). CSE activity was assessed bycystathionine consumption and enzyme activity expressed as fold changecalculated from nanomoles of cystathionine consumed per mg of totalprotein per hour of incubation.

2.10 Western blot analysis: Mouse skeletal muscle tissues werehomogenized in a solution containing 50 mM Tris buffer (pH 7.4), 2 mMEDTA, 5 mM EGTA, 0.1% SDS, a protease inhibitor cocktail (Roche,Indianapolis, IN), and phosphatase inhibitor cocktail type I and II(Sigma, Saint Louis, MO). Homogenates were centrifuged at 500×g for 15min and supernatants were collected. Protein concentrations wereanalyzed using the Bradford protein assay (BIORAD, Hercules, CA).Proteins were separated using 10% SDS-PAGE (Bio-Rad, Hercules, CA) andtransferred onto PVDF membranes, and incubated with antibodies againstCSE #12217-1-AP (Fisher Scientific), eNOS #9572, phospho-eNOS #9750 andGAPDH #2118 (Cell Signaling). Chemiluminescent bands were detected andquantified using NIH Image J software.

2.11 Quantitative PCR: Samples were stored in TRIzol reagent (ThermoFisher Scientific Inc., Waltham, MA, USA) and RNA was isolated usingphenol:chloroform extraction procedure. RNA concentration and puritywere evaluated with a NanoDrop 2000 spectrometer (Thermo FisherScientific Inc., Waltham, MA, USA). RNA samples with an absorbance ratioOD 260/280 between 1.8-2.1 and OD 260/230 between 2-2.2 were used forfurther analysis. Single-stranded cDNA was synthesized using ISCRIPTcDNA synthesis kit (Bio-Rad, Hercules, CA, USA), from 1 μg of total RNAin a final volume of 20 μL. cDNA was stored at −20° C. for future use.Quantitative PCR reactions were performed using the universal SYBR GreenSupermix (Bio-Rad, CA, USA) on a CFX96 thermal cycler with Bio-Rad CFXManager software (Bio-Rad, Hercules, CA, USA). 50 ng of cDNA were usedfor each reaction. The reactions for each sample were run in triplicate.The mean threshold cycle (Ct) values for each serial dilution wasplotted against the logarithm of the cDNA dilution factor.

2.11 Immunohistochemistry: Immunohistochemistry staining with anti-CD31and anti-CSE antibodies was performed with nuclear stain4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI), as the inventorshave previously described.

2.12 Oxidative Stress measurement: Levels of oxidative stress weremeasured by staining skeletal muscle tissue sections with 5 μMDihydroethidium (DHE; Sigma-Aldrich, CA USA) and visualized using aNIKON ECLIPSE Ti-E microscope (Nikon Instruments Inc., Melville, NY) forimage acquisition. Simple PCI software version 6.0 (Compix Inc.,Sewickley, PA, USA) was used to analyze the area.

2.12 Statistical Analysis: Data were reported as mean±standard error ofthe mean (SEM) for all groups. Statistical analysis was performed withGraphPad Prism using Student's t-test, one-way ANOVA and two-way ANOVAwith Tukey post-hoc test, Mann-Whitney or Kruskal-Wallis analysis ofvariance with Dunn's multiple-comparison tests. A p-value of <0.05 wasconsidered to be statistically significant.

Results:

Methamphetamine experimental ‘binge and crash’ model causes endothelialcell dysfunction. In the systemic circulation, terminal arteries arecrucial for regulating blood flow to all organs throughout the body.Moreover, arteries and blood vessels are significantly affected bymethamphetamine use resulting in cardiovascular dysfunction and centralnervous system damage. Methamphetamine induces vasoconstriction thatreduces essential blood flow, increases blood pressure and bloodclotting, which can lead to increased bleeding in the brain and strokeand may cause abnormalities in the heart. the inventors have used anon-invasive flow mediated dilation (FMD) model in mice to assessendothelial function of femoral arteries at the end of the 4-week ‘bingeand crash’ methamphetamine treatment protocol. The mouse FMD modelentails temporary 1-minute occlusion of limb blood flow using aninflatable cuff followed by doppler ultrasound measurement of changes inblood flow following release of the cuff. Real time measurements ofvascular diameter changes and mean blood flow velocity are collectedreflecting changes in endovascular function and health. FIGS. 6A and 6Bshow reactive doppler blood flow responses between saline control versusmethamphetamine treated mice, respectively. FIG. 6C shows thatmethamphetamine treated mice have significantly blunted flow mediatedvasodilation responses at (what timepoint?) compared to saline treatedcontrol mice. Lastly, panel 6D illustrates that methamphetaminetreatment significantly blunts mean blood flow velocity recovery afterocclusion removal. These data show that methamphetamine elicitsendothelial dysfunction in response to flow mediated vasodilation.

Methamphetamine ‘binge and crash’ decreases hydrogen sulfide and nitricoxide bioavailability. Plasma NO and H₂S bioavailability are associatedwith endothelial and vascular dysfunction. Therefore, plasma and tissuemetabolites of NO and H₂S were measured at the end of the 4-week ‘bingeand crash’ model of methamphetamine treatment. A significant reductionin sulfide metabolites-acid-labile and bound sulfane sulfur pools can beseen with methamphetamine compared to controls (FIGS. 1A and B).Similarly, NO metabolites—free nitrite and bound pool, S-nitrosothiolwere decreased in methamphetamine treated group (FIGS. 1D and 1E).Methamphetamine causes a 3-fold reduction in plasma total sulfide (FIG.1C) and a two-fold reduction in plasma total NO levels (FIG. 1F).Subsequently, in the skeletal muscle tissues a significant decrease intotal sulfide levels were observed in mice treated with methamphetamine.Similarly, total NO in the skeletal muscles was significantly reduced inmethamphetamine treated mice. These data evidence a role of H₂Sgeneration pathways (CSE, cystathionine beta synthase (CBS) or 3-MST)that can maintain the muscle tissue total sulfide levels in themethamphetamine treated mice. As mentioned above, CSE dependent H₂Sproduction regulates NO bioavailability under basal and ischemic states.This indicates that methamphetamine treatment significantly reduces H₂Sand NO metabolites, which is associated with endothelial dysfunction.

Methamphetamine ‘binge and crash’ blunts CSE expression and increasesinflammation. H₂S is predominantly produced by CSE and CBS enzymes inthe vasculature and other tissues, whereas NO bioavailability isenzymatically influenced by eNOS. To check if compensation in sulfidelevels was mediated by enzymatic mechanisms, the inventors measured theexpressions of H₂S producing enzymes, CSE and CBS in methamphetaminemice. Methamphetamine significantly decreased CSE gene expression (FIG.2A), however, no significant changes were observed in gene expression ofCBS in skeletal muscle tissue (FIG. 2B). Interestingly, no change ineNOS expression was observed, although there were decreased NO levels inmethamphetamine treated mice (FIG. 2C). Posttranslational modificationssuch as phosphorylation of eNOS can influence the bioavailability of NO.So, the inventors further looked at the protein expressions of CSE,p-eNOS and total eNOS (FIG. 2D). Methamphetamine treated mice skeletalmuscle showed decreased expressions of CSE and p-eNOS, quantified inFIGS. 2E and F; however, no change in total eNOS was observed.

Methamphetamine blunts CSE expression in mouse and human tissues.Further to the inventors' observations of reduced CSE mRNA levels, theinventors examined CSE protein expression in sections of mouse skeletalmuscle tissue from the ‘binge and crash’ model and human heart leftventricular (LV) tissues of methamphetamine users (FIGS. 3 and 10 ). Theinventors found a significant decrease in vascular CSE expression inskeletal muscles of methamphetamine-treated mice compared tosaline-treated controls (panels 3B vs 3A, respectively). Likewise, theinventors also found a significant reduction in vascular CSE expression(as seen by reduced co-localization with the endothelial marker CD31) inhearts collected post-mortem from human methamphetamine-users, comparedto non-methamphetamine using individuals (panels 10B vs 10A,respectively). Semi-quantitative image analysis revealed thatmethamphetamine treatment in mice and methamphetamine use in humanssignificantly decreases CSE protein expression (panels 3C and 10C,respectively).

Methamphetamine ‘binge and crash’ increases skeletal tissue oxidativestress. FIG. 5 shows methamphetamine-mediated increase in oxidativestress using the fluorescent probe for superoxide, dihydrohydroethidine(DHE) in stained sections of skeletal muscle tissues treated with salineor methamphetamine, respectively. There is a significant increase in DHEfluorescence indicating an increase in oxidative stress production withmethamphetamine treatments (FIGS. 5A and 5B). Chronic methamphetamineuse can induce neuro- and cardio-toxicity due to increased oxidativestress and pro-inflammatory cytokines including ICAM-1, VCAM-1, TNF-α,and IFN-γexpression that promotes atherosclerosis. H₂S counteractsoxidative stress under chronic conditions through upregulation ofantioxidant defenses. It also inhibits leukocyte-endothelial cellinteractions in response to acute ischemia/reperfusion injury indicatinganti-inflammatory action preventing endothelial cell activation. Aconcomitant increase in vascular cell adhesion molecule (VCAM-1) andintercellular adhesion molecule (ICAM-1) expression is observed withmethamphetamine treatment (panels 5D and 5E). NADPH oxidases (NOX) aremajor sources of oxidative stress. NOx4 has a predominant role inregulating oxidative stress under ischemic stress conditions and in thecardiovascular system. the inventors observed a marginal increase inNOx4 expression with methamphetamine treatment in skeletal muscles (FIG.5F). This indicates that increased oxidative stress and pro-inflammatoryphenotype can contribute to alterations of vascular tone and blood flow.

Methamphetamine ‘binge and crash’ decreases skeletal tissue CSE proteinand endothelial CSE enzyme activity. Next, the inventors examined theeffect of methamphetamine treatment (100 μM) on CSE enzyme activity inendothelial cells, which are a major component of vascular bloodvessels. the inventors found that methamphetamine treatment of HUVECstreated for 30 min substantially decreased CSE enzyme activity over timecompared to PBS control treatment (FIG. 11A). Apart from the MAECs,significant decrease in CSE activity was observed in skeletal muscletissues of methamphetamine treated mice (FIG. 4A), and plasma from humanmethamphetamine-users compared to non-users (FIG. 11B).

CSE or exogenous sulfide rescues methamphetamine inhibited vasculardilation via sulfide. To check whether rescue of vascular function canbe rectified via upregulating CSE or sulfide bioavailability, theinventors used WT mice with parallel treatments of sulfide or CSE Tgmice along with methamphetamine treatments. the inventors observed asignificant recovery in endothelial function via femoral vasodilationwith FMD model via sulfide therapy or CSE overexpression (FIG. 7A). Asignificant recovery (25 and 30% respectively) was observed in mean flowvelocity in methamphetamine treated mice with simultaneous sulfidetherapy or in CSE Tg mice, as shown in FIG. 7B. Additionally, theinventors observed an increase in plasma total sulfide levels andthree-fold increase in skeletal muscle tissues compared to bothmethamphetamine treated and control mice (results not shown). To checkwhether this therapy has any effect on CSE expression in vascularsystem, the inventors observed a two-fold increase in CSE activity infemoral arteries. This evidences that each of sulfide therapy and CSEoverexpression rescues vascular function.

CSE/Sulfide corrects pro-aging effects of methamphetamine:. Aging is anindependent predictor of cardiovascular complications. Advancing agepromotes cardiovascular disease (CVD), ultimately leading to death.Aging and subsequent complications includes artery stiffening andendothelial dysfunction, which are responsible for the development ofCVD. A previous study indicates that transcription factor, ATF4, caninduce CSE expression. So, the inventors checked for changes inage-related genes, Sirtuins (Sirt), including ATF4 and CSE in tissues ofmethamphetamine treated mice. A significant increase in CSE and ATF4expressions (FIGS. 9A and 9B, respectively) were observed with sulfidetherapy and CSETg mice with methamphetamine treatments. Similarly, aminimum of a two-fold increase in expressions of Sirt1 and Sirt6 (FIGS.9C and 9D, respectively) were also observed, compared to methamphetaminealone treated controls. This is evidence that the pro-aging effects ofmethamphetamine could partially be protected with exogenous sulfide oroverexpression of CSE.

Discussion:

Understanding of methamphetamine-related cardiovascular implications andthe underlying molecular mechanisms remain poorly understood in currenttechnology. Methamphetamine can have potentially fatal effects oncardiovascular pathology including atherosclerosis, which ischaracterized by plaque formation and occlusion of the blood vesselsleading to increased morbidity and mortality. The development ofocclusions in peripheral arteries in a young methamphetamine use subjectare evidence of fatal effects of methamphetamine-mediated vascularpathology.

Decreased cerebral blood flow is observed through sustainedmethamphetamine-induced vasoconstriction of pial arterioles. Endothelialdysfunction is the primary cause for much cardiovascular pathology.Methamphetamine can cause severe endothelial dysfunction leading toincreased atherosclerosis. In this disclosure, the inventors demonstratemethamphetamine mediated endovascular dysfunction and the underlyingmolecular mechanisms that regulate them. Results from FMD show thatmethamphetamine induces constriction in the femoral artery and decreasesthe mean blood velocity, resulting in dysfunctional vascular tone anddefective blood flow. The gasotransmitters NO and H₂S play keyregulatory roles in many pathophysiological functions. Both NO—H₂Ssignaling pathway crosstalk to mediate their effects on vascularfunctions, including vasodilation and vascular remodeling. theinventors' results indicate that defect in vasodilation is associatedwith significant reduction H₂S and NO bioavailability in plasmas frommethamphetamine-treated mice. To further check if these changes incirculation were reflected in the tissues, the inventors checked thesulfide and NO levels in the skeletal muscle tissues. the inventorsobserved no significant changes in total sulfide levels, however, totalNO levels were significantly decreased compared to the saline controls.H₂S regulates NO metabolic pathways during ischemic vascular remodeling,which is reflected in patients with clinical vascular disease. Similarto these observations, the inventors saw a decrease in acid-labilesulfide levels in human plasma samples from methamphetamine-userscompared to age matched healthy controls, however, there is a trend ofdecreasing total NO levels with no statistical significance.

Cystathionine γ-lyase (CSE) is a major enzyme producing H₂S in thevascular system that plays critical roles in endothelial function andcardiovascular health. However, H₂S can also be synthesized by CBS,another enzyme in the transsulfuration pathway. CSE dependent H₂Sproduction is critically important for regulation of vascular functionand remodeling. Compensation of H₂S bioavailability in the skeletalmuscle can be possible via increased CBS expression. Interestingly, CSEgene and protein expressions were significantly reduced withmethamphetamine treatment in skeletal muscle tissues (4A and D). Nostatistically significant compensation was observed in CBS or changes intotal eNOS expression, though this may have been due to the small samplesize. It is noteworthy to see a decrease in eNOS phosphorylation atSer1177, in skeletal muscle tissues of methamphetamine-treated micecompared to saline controls. This evidences that methamphetaminesignificantly inhibits CSE and p-eNOS expressions thereby decreasing H₂Sand NO bioavailability in mice. Likewise, immunohistology of skeletalmuscle tissues shows a decrease in CSE expression in methamphetaminetissue compared to saline controls. Similarly, a significant decrease inCSE activity was observed in endothelial cells and skeletal muscle ofmice treated with methamphetamine. Corroborating these observations, LVheart sections from humans revealed that methamphetamine users have asignificant reduction in CSE compared to non-methamphetamine controls.This corresponds with the decrease in CSE activity inmethamphetamine-treated endothelial cells, mouse skeletal muscle tissuesand in plasma of human methamphetamine-users. These results evidencethat methamphetamine treatment can critically inhibit CSE expression andsubsequent H₂S bioavailability.

Methamphetamine can induce oxidative stress and ROS production that canlead to long-lasting damage to neurological system. Enhanced oxidativestress represent common effects of methamphetamine use; specific reasonsfor these molecular changes underlying much of the cardiovascularcomplications are still not known completely. Methamphetamine-mediatedincrease in oxidative stress using the fluorescent probe for superoxide,dihydrohydroethidine (DHE) in stained sections of skeletal muscletissues of mice treated with saline or methamphetamine, respectively.There was a significant increase in DHE fluorescence indicating anincrease in oxidative stress production with methamphetamine treatments.These results substantiate the inventors' observations thatmethamphetamine increases oxidative stress and may lead tocardiovascular dysfunction. Cellular adhesion molecules, VCAM-1 andICAM-1 play important roles in transendothelial migration of leukocytes,and within the milieu of the atherosclerotic plaque. Circulating ICAM-1,VCAM-1 have been detected in plasma and are elevated during inflammatoryconditions in the prediction of cardiovascular disease. Chronicmethamphetamine use can induce neuro- and cardio-toxicity due toincreased oxidative stress and pro-inflammatory cytokines includingICAM-1, VCAM-1, TNF-α, and IFN-γ expression that promotesatherosclerosis. H₂S counteracts oxidative stress under chronicconditions through upregulation of antioxidant defenses. It has alsobeen shown to inhibit leukocyte-endothelial cell interactions inresponse to acute ischemia/reperfusion injury indicatinganti-inflammatory action preventing endothelial cell activation. Theinventors showed that methamphetamine significantly decreased CSE butnot CBS gene expression in skeletal muscle tissue. Importantly, aconcomitant increase in ICAM-1 and VCAM-1 expression is observed withmethamphetamine treatment. NADPH oxidases (NOX) are major sources ofoxidative stress. Nox4 has a predominant role in regulating oxidativestress under ischemic stress conditions and in the cardiovascularsystem. Furthermore, recent literature implicates a role for NOX inmethamphetamine-induced oxidative stress in brain endothelial cells.However, the role of Nox4 in methamphetamine-induced oxidative stressand regulation of vasomotor activity in the vasculature requires furtherinvestigation. A trend of increasing NOX4 gene expression can beobserved in skeletal muscle tissues of methamphetamine-treated mice.These data reveal that loss of CSE gene expression is associated withincreased pro-inflammatory activation in the vasculature.

Defects in CSE and H₂S critically impair vascular function, includingvasodilation and vessel remodeling. Results from FMD indicate adysfunctional vascular tone, femoral artery constriction and decreasedblood flow due to methamphetamine-mediated CSE/H₂S inhibition. Severeimpairment of CSE/H₂S leads to significant reduction in plasma andtissue NO levels and tissue growth factor expression resulting inimpaired revascularization and blood flow responses, which can berectified by exogenous H₂S based donor therapy. the inventors increasedthe CSE/H₂S levels through exogenous or endogenous delivery to rectifymethamphetamine-mediated vascular defects, via simultaneous treatmentsof methamphetamine and exogenous treatment with Na₂S in C57BL6/J mice orin CSE Tg mice. FMD analyses of these mice groups showed a significantrecovery in the vessel dilation and blood flow velocities, which isassociated with concurrent elevation of H₂S bioavailability in plasmaand skeletal muscle and blood vessel CSE activity in femoral arteries.This further corroborates the inventors' observations that endogenousCSE/H₂S regulates endothelial and vascular function and rescuesmethamphetamine-inhibited vascular tone.

In the current disclosure, the inventors demonstrate thatmethamphetamine induces endothelial dysfunction via CSE inhibition andsubsequent reduction of H₂S bioavailability. the inventors' resultsreveal for the first time that a methamphetamine mediated decrease inCSE expression and activity can contribute to vascular dysfunctionincluding increased oxidative stress, inflammatory activation, andreduced H₂S/NO bioavailability in mouse models. However, the inventorsonly observed a decrease in acid-labile sulfide pools in humanmethamphetamine-users. There could be many possibilities due to durationof the methamphetamine and how long the user had been offmethamphetamine usage.

Methamphetamine use has effects on vascular dysfunction and oncirculating metabolites of H₂S/NO that regulate critical functions ofcardiovascular pathophysiology. the inventors' observations clearlydemonstrated that methamphetamine severely dysregulates normal vasculartone of the femoral artery, which can be rectified byexogenous/endogenous CSE/H₂S therapy. Collectively, the inventors'results reveal for the first time that methamphetamine inhibits CSEvascular expression and activity in endothelial cells that contribute tovascular dysfunction. Methamphetamine use leads to atherosclerosis andassociated complications, thereby contribute to increased cardiovasculardisease. the inventors' study has revealed important insights intomethamphetamine-mediated vascular dysfunction and associated molecularsignaling, highlighting clinical importance of CSE/H₂S role, as atherapeutic targets.

Turning to FIGS. 12-13C, the results of experiments with thepharmaceutical Sugammadex are shown, monitoring sulfide levels in wildtype and CSE knockout mice (FIG. 12 ) and demonstrating that it alsoincreases human bound sulfane sulfur and total sulfide levels in humanblood (FIGS. 13A and 13B) along with increased plasma CSE activity (FIG.13C).

Sugammadex is a γ-cyclodextrin medication for the reversal ofneuromuscular blockade induced by rocuronium and vecuronium in generalanesthesia. It has a molecular formula of C₇₂H₁₀₄Na₈O₄₈S₈, and achemical structure of:

In clinical practice, sugammadex is administered at a dose of 4 mg/kg toup to 16 mg/kg for its current use. In the inventors' mice studies, theinventors used a single injection of 0.2 mg/kg for pharmacokineticstudies, which was a 20 to 80 times lower than the clinical usage ofsugammadex.

For initial experiments disclosed herein, Sulfide and DATSconcentrations in drinking water were used at 30 uM at an average of 3-4mls of water intake. The inventors have previously used sulfide and DATSintravenously at 200 ug/kg concentration twice daily in mice withchronic ischemia.

Pharmaceutical Compositions:

The methods described herein can also include the administrations ofpharmaceutically acceptable compositions that include the therapeutic,or a pharmaceutically acceptable salt, solvate, or prodrug thereof. Whenemployed as pharmaceuticals, any of the present compounds can beadministered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical, parenteral,intravenous, intra-arterial, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration.

This invention also includes pharmaceutical compositions which cancontain one or more pharmaceutically acceptable carriers. In making thepharmaceutical compositions of the invention, the active ingredient istypically mixed with an excipient, diluted by an excipient or enclosedwithin such a carrier in the form of, for example, a capsule, sachet,paper, or other container. When the excipient serves as a diluent, itcan be a solid, semisolid, or liquid material (e.g., normal saline),which acts as a vehicle, carrier or medium for the active ingredient.Thus, the compositions can be in the form of tablets, powders, lozenges,sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups,and soft and hard gelatin capsules. As is known in the art, the type ofdiluent can vary depending upon the intended route of administration.The resulting compositions can include additional agents, such aspreservatives.

The therapeutic agents of the invention can be administered alone, or ina mixture, in the presence of a pharmaceutically acceptable excipient orcarrier. The excipient or carrier is selected on the basis of the modeand route of administration. Suitable pharmaceutical carriers, as wellas pharmaceutical necessities for use in pharmaceutical formulations,are described in Remington: The Science and Practice of Pharmacy,22^(nd) Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2012), awell-known reference text in this field, and in the USP/NF (UnitedStates Pharmacopeia and the National Formulary), each of which isincorporated by reference. In preparing a formulation, the activecompound can be milled to provide the appropriate particle size prior tocombining with the other ingredients. If the active compound issubstantially insoluble, it can be milled to a particle size of lessthan 200 mesh. If the active compound is substantially water soluble,the particle size can be adjusted by milling to provide a substantiallyuniform distribution in the formulation, e.g., about 40 mesh.

Examples of suitable excipients are lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Otherexemplary excipients are described in Handbook of PharmaceuticalExcipients, 8^(th) Edition, Sheskey et al., Eds., Pharmaceutical Press(2017), which is incorporated by reference.

The methods described herein can include the administration of atherapeutic, or prodrugs or pharmaceutical compositions thereof, orother therapeutic agents.

The pharmaceutical compositions can be formulated so as to provideimmediate, extended, or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining, e.g., 0.1-500 mg of the active ingredient. For example, thedosages can contain from about 0.1 mg to about 50 mg, from about 0.1 mgto about 40 mg, from about 0.1 mg to about 20 mg, from about mg to about10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg;from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg,from about 0.5 mg to about 30 mg, from about 0.5 mg to about 20 mg, fromabout 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about1 mg from to about 50 mg, from about 1 mg to about 30 mg, from about 1mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg toabout 5 mg; from about 5 mg to about mg, from about 5 mg to about 20 mg,from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, fromabout 20 mg to about 200 mg, from about 30 mg to about 150 mg, fromabout mg to about 100 mg, from about 50 mg to about 100 mg of the activeingredient, from about mg to about 300 mg, from about 50 mg to about 250mg, from about 100 mg to about 300 mg, or, from about 100 mg to about250 mg of the active ingredient. For preparing solid compositions suchas tablets, the principal active ingredient is mixed with one or morepharmaceutical excipients to form a solid bulk formulation compositioncontaining a homogeneous mixture of a compound of the present invention.When referring to these bulk formulation compositions as homogeneous,the active ingredient is typically dispersed evenly throughout thecomposition so that the composition can be readily subdivided intoequally effective unit dosage forms such as tablets and capsules. Thissolid bulk formulation is then subdivided into unit dosage forms of thetype described above containing from, for example, 0.1 to about 500 mgof the active ingredient of the present invention.

Compositions for Oral Administration: The pharmaceutical compositionscontemplated by the invention include those formulated for oraladministration (“oral dosage forms”). Oral dosage forms can be, forexample, in the form of tablets, capsules, a liquid solution orsuspension, a powder, or liquid or solid crystals, which contain theactive ingredient(s) in a mixture with non-toxic pharmaceuticallyacceptable excipients. These excipients may be, for example, inertdiluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol,microcrystalline cellulose, starches including potato starch, calciumcarbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate,or sodium phosphate); granulating and disintegrating agents (e.g.,cellulose derivatives including microcrystalline cellulose, starchesincluding potato starch, croscarmellose sodium, alginates, or alginicacid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginicacid, sodium alginate, gelatin, starch, pregelatinized starch,microcrystalline cellulose, magnesium aluminum silicate,carboxymethylcellulose sodium, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethyleneglycol); and lubricating agents, glidants, and antiadhesives (e.g.,magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenatedvegetable oils, or talc). Other pharmaceutically acceptable excipientscan be colorants, flavoring agents, plasticizers, humectants, bufferingagents, and the like.

Formulations for oral administration may also be presented as chewabletablets, as hard gelatin capsules wherein the active ingredient is mixedwith an inert solid diluent (e.g., potato starch, lactose,microcrystalline cellulose, calcium carbonate, calcium phosphate orkaolin), or as soft gelatin capsules wherein the active ingredient ismixed with water or an oil medium, for example, peanut oil, liquidparaffin, or olive oil. Powders, granulates, and pellets may be preparedusing the ingredients mentioned above under tablets and capsules in aconventional manner using, e.g., a mixer, a fluid bed apparatus or aspray drying equipment.

Controlled release compositions for oral use may be constructed torelease the active drug by controlling the dissolution and/or thediffusion of the active drug substance. Any of a number of strategiescan be pursued in order to obtain controlled release and the targetedplasma concentration vs time profile. In one example, controlled releaseis obtained by appropriate selection of various formulation parametersand ingredients, including, e.g., various types of controlled releasecompositions and coatings. Thus, the drug is formulated with appropriateexcipients into a pharmaceutical composition that, upon administration,releases the drug in a controlled manner. Examples include single ormultiple unit tablet or capsule compositions, oil solutions,suspensions, emulsions, microcapsules, microspheres, nanoparticles,patches, and liposomes. In certain embodiments, compositions includebiodegradable, pH, and/or temperature-sensitive polymer coatings.

Dissolution or diffusion-controlled release can be achieved byappropriate coating of a tablet, capsule, pellet, or granulateformulation of compounds, or by incorporating the compound into anappropriate matrix. A controlled release coating may include one or moreof the coating substances mentioned above and/or, e.g., shellac,beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glycerylmonostearate, glyceryl distearate, glycerol palmitostearate,ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetatebutyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone,polyethylene, polymethacrylate, methylmethacrylate,2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol,ethylene glycol methacrylate, and/or polyethylene glycols. In acontrolled release matrix formulation, the matrix material may alsoinclude, e.g., hydrated methylcellulose, carnauba wax and stearylalcohol, carbopol 934, silicone, glyceryl tristearate, methylacrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/orhalogenated fluorocarbon.

The liquid forms in which the compounds and compositions of the presentinvention can be incorporated for administration orally include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

Compositions suitable for oral mucosal administration (e.g., buccal orsublingual administration) include tablets, lozenges, and pastilles,where the active ingredient is formulated with a carrier, such as sugar,acacia, tragacanth, or gelatin and glycerin.

Coatings: The pharmaceutical compositions formulated for oral delivery,such as tablets or capsules of the present invention can be coated orotherwise compounded to provide a dosage form affording the advantage ofdelayed or extended release. The coating may be adapted to release theactive drug substance in a predetermined pattern (e.g., in order toachieve a controlled release formulation) or it may be adapted not torelease the active drug substance until after passage of the stomach,e.g., by use of an enteric coating (e.g., polymers that are pH-sensitive(“pH controlled release”), polymers with a slow or pH-dependent rate ofswelling, dissolution or erosion (“time-controlled release”), polymersthat are degraded by enzymes (“enzyme-controlled release” or“biodegradable release”) and polymers that form firm layers that aredestroyed by an increase in pressure (“pressure-controlled release”)).Exemplary enteric coatings that can be used in the pharmaceuticalcompositions described herein include sugar coatings, film coatings(e.g., based on hydroxypropyl methylcellulose, methylcellulose, methylhydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose,acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone),or coatings based on methacrylic acid copolymer, cellulose acetatephthalate, hydroxypropyl methylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, polyvinyl acetate phthalate, shellac,and/or ethylcellulose. Furthermore, a time delay material such as, forexample, glyceryl monostearate or glyceryl distearate, may be employed.

For example, the tablet or capsule can comprise an inner dosage and anouter dosage component, the latter being in the form of an envelope overthe former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. When an enteric coating is used, desirably, a substantialamount of the drug is released in the lower gastrointestinal tract.

In addition to coatings that effect delayed or extended release, thesolid tablet compositions may include a coating adapted to protect thecomposition from unwanted chemical changes (e.g., chemical degradationprior to the release of the active drug substance). The coating may beapplied on the solid dosage form in a similar manner as that describedin Encyclopedia of Pharmaceutical Technology, vols. 5 and 6, Eds.Swarbrick and Boyland, 2000.

Parenteral Administration: Within the scope of the present invention arealso parenteral depot systems from biodegradable polymers. These systemsare injected or implanted into the muscle or subcutaneous tissue andrelease the incorporated drug over extended periods of time, rangingfrom several days to several months. Both the characteristics of thepolymer and the structure of the device can control the release kineticswhich can be either continuous or pulsatile. Polymer-based parenteraldepot systems can be classified as implants or microparticles. Theformer are cylindrical devices injected into the subcutaneous tissuewhereas the latter are defined as spherical particles in the range of10-100 μm. Extrusion, compression or injection molding are used tomanufacture implants whereas for microparticles, the phase separationmethod, the spray-drying technique and the water-in-oil-in-wateremulsion techniques are frequently employed. The most commonly usedbiodegradable polymers to form microparticles are polyesters from lacticand/or glycolic acid, e.g., poly(glycolic acid) and poly(L-lactic acid)(PLG/PLA microspheres). Of particular interest are in situ forming depotsystems, such as thermoplastic pastes and gelling systems formed bysolidification, by cooling, or due to the sol-gel transition,cross-linking systems and organogels formed by amphiphilic lipids.Examples of thermosensitive polymers used in the aforementioned systemsinclude, N-isopropylacrylamide, poloxamers (ethylene oxide and propyleneoxide block copolymers, such as poloxamer 188 and 407), poly(N-vinylcaprolactam), poly(siloethylene glycol), polyphosphazenes derivativesand PLGA-PEG-PLGA.

Mucosal Drug Delivery: Mucosal drug delivery (e.g., drug delivery viathe mucosal linings of the nasal, rectal, vaginal, ocular, or oralcavities) can also be used in the methods described herein. Methods fororal mucosal drug delivery include sublingual administration (viamucosal membranes lining the floor of the mouth), buccal administration(via mucosal membranes lining the cheeks), and local delivery (Harris etal., Journal of Pharmaceutical Sciences, 81(1): 1-10, 1992).

Oral transmucosal absorption is generally rapid because of the richvascular supply to the mucosa and allows for a rapid rise in bloodconcentrations of the therapeutic.

For buccal administration, the compositions may take the form of, e.g.,tablets, lozenges, etc. formulated in a conventional manner. Permeationenhancers can also be used in buccal drug delivery. Exemplary enhancersinclude 23-lauryl ether, aprotinin, azone, benzalkonium chloride,cetylpyridinium chloride, cetyltrimethylammonium bromide, cyclodextrin,dextran sulfate, lauric acid, lysophosphatidylcholine, methol,methoxysalicylate, methyloleate, oleic acid, phosphatidylcholine,polyoxyethylene, polysorbate 80, sodium EDTA, sodium glycholate, sodiumglycodeoxycholate, sodium lauryl sulfate, sodium salicylate, sodiumtaurocholate, sodium taurodeoxycholate, sulfoxides, and alkylglycosides. Bioadhesive polymers have extensively been employed inbuccal drug delivery systems and include cyanoacrylate, polyacrylicacid, hydroxypropyl methylcellulose, and poly methacrylate polymers, aswell as hyaluronic acid and chitosan.

Liquid drug formulations (e.g., suitable for use with nebulizers andliquid spray devices and electrohydrodynamic (EHD) aerosol devices) canalso be used. Other methods of formulating liquid drug solutions orsuspension suitable for use in aerosol devices are known to those ofskill in the art (see, e.g., Biesalski, U.S. Pat. No. 5,112,598, andBiesalski, U.S. Pat. No. 5,556,611).

Formulations for sublingual administration can also be used, includingpowders and aerosol formulations. Exemplary formulations include rapidlydisintegrating tablets and liquid-filled soft gelatin capsules.

The pharmaceutical compositions of the invention may be dispensed to thesubject under treatment with the help of an applicator. The applicatorto be used may depend on the specific medical condition being treated,amount and physical status of the pharmaceutical composition, and choiceof those skilled in the art. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be employed.In certain applications, an ointment, lotion, cream, gel or similarformulation can be provided that can be applied to the skin using thefingers. Such formulations are typically provided in a squeeze tube orbottle or a pot, or in a roll-on, wherein a ball is secured in the topof a container of the formulation, wherein the ball is permitted toroll. By rolling the ball over the skin surface, liquid in the containeris transferred to the skin in a controlled manner. An alternativedelivery mechanism includes a container with a perforated lid with amechanism for advancing an extrudable formulation through the lid. Inanother form, a gel formulation with sufficient structural integrity tomaintain its shape is provided, which is advanced up a tube and appliedto the skin (e.g., in a stick form). An advantage of the stick form isthat only the formulation contacts the skin in the application process,not the fingers or a portion of a container. A liquid or gel can also beplaced using an applicator, e.g., a wand, a sponge, a syringe, or othersuitable method.

The pharmaceutical compositions of the invention may be provided to thesubject or the medical professional in charge of dispensing thecomposition to the subject, along with instructional material. Theinstructional material includes a publication, a recording, a diagram,or any other medium of expression, which may be used to communicate theusefulness of the composition and/or compound used in the practice ofthe invention in a kit. The instructional material of the kit may, forexample, be affixed to a container that contains the compound and/orcomposition used in the practice of the invention or shipped togetherwith a container that contains the compound and/or composition.Alternatively, the instructional material may be shipped separately fromthe container with the intention that the recipient uses theinstructional material and the compound cooperatively. Delivery of theinstructional material may be, for example, by physical delivery of thepublication or other medium of expression communicating the usefulnessof the kit, or may alternatively be achieved by electronic transmission,for example by means of a computer, such as by electronic mail, ordownload from a website.

Other routes of administration to the affected area which arecontemplated include: transdermal, mucosal, rectal, and vaginal, ortopical (for example, in a carrier vehicle, a topical control releasepatch, in a wound dressing, a hydrocolloid, a foam, or a hydrogel, acream, a gel, a lotion, an ointment, a liquid crystal emulsion (LCE),and/or a micro-emulsion). An appropriate biological carrier orpharmaceutically acceptable excipient may be used. Compoundsadministered may, in various embodiments, be racemic, isomericallypurified, or isomerically pure.

Transmucosal Administration: Transmucosal administration is carried outusing any type of formulation or dosage unit suitable for application tomucosal tissue. For example, the selected active agent may beadministered to the buccal mucosa in an adhesive tablet or patch,sublingually administered by placing a solid dosage form under thetongue, lingually administered by placing a solid dosage form on thetongue, administered nasally as droplets or a nasal spray, a non-aerosolliquid formulation, or a dry powder, placed within or near the rectum(“transrectal” formulations), or administered to the urethra as asuppository, ointment, or the like. Application in the oral or nasalcavities are options for high absorption that does not make a first passin the liver.

Transrectal Administration: Transrectal dosage forms may include rectalsuppositories, creams, ointments, and liquid formulations (enemas). Thesuppository, cream, ointment, or liquid formulation for transrectaldelivery comprises a therapeutically effective amount of the selectedactive agent and one or more conventional nontoxic carriers suitable fortransrectal drug administration. The transrectal dosage forms of thepresent invention may be manufactured using conventional processes. Thetransrectal dosage unit may be fabricated to disintegrate rapidly orover a period of several hours. The time period for completedisintegration may be in the range of from about 10 minutes to about 6hours, e.g., less than about 3 hours. This can be an option foradministration for high absorption that does not make a first pass inthe liver.

Vaginal or Perivaginal Administration. Vaginal or perivaginal dosageforms may include vaginal suppositories, creams, ointments, liquidformulations, pessaries, tampons, gels, pastes, foams, or sprays. Thesuppository, cream, ointment, liquid formulation, pessary, tampon, gel,paste, foam, or spray for vaginal or perivaginal delivery comprises atherapeutically effective amount of the selected active agent and one ormore conventional nontoxic carriers suitable for vaginal or perivaginaldrug administration. The vaginal or perivaginal forms of the presentinvention may be manufactured using conventional processes as disclosedin Remington: The Science and Practice of Pharmacy, supra (see also drugformulations as adapted in U.S. Pat. Nos. 6,515,198; 6,500,822;6,417,186; 6,416,779; 6,376,500; 6,355,641; 6,258,819; 6,172,062; and6,086,909). The vaginal or perivaginal dosage unit may be fabricated todisintegrate rapidly or over a period of several hours. The time periodfor complete disintegration may be in the range of from about 10 minutesto about 6 hours, e.g., less than about 3 hours. This can be an optionfor administration for high absorption that does not make a first passin the liver.

Topical Formulations: Topical formulations may be in any form suitablefor application to the body surface, and may comprise, for example, anointment, cream, gel, lotion, solution, paste or the like, and/or may beprepared so as to contain liposomes, micelles, and/or microspheres. Incertain embodiments, topical formulations herein are ointments, creams,and gels.

Transdermal Administration: Transdermal compound administration, whichis known to one skilled in the art, involves the delivery ofpharmaceutical compounds via percutaneous passage of the compound intothe systemic circulation of the patient. Topical administration may alsoinvolve the use of transdermal administration such as transdermalpatches or iontophoresis devices. Other components may be incorporatedinto the transdermal patches as well. For example, compositions and/ortransdermal patches may be formulated with one or more preservatives orbacteriostatic agents including, but not limited to, methylhydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkoniumchloride, and the like. Dosage forms for topical administration of thecompounds and compositions may include creams, sprays, lotions, gels,ointments, eye drops, nose drops, ear drops, and the like. In suchdosage forms, the compositions of the invention may be mixed to formwhite, smooth, homogeneous, opaque cream or lotion with, for example,benzyl alcohol 1% or 2% (wt/wt) as a preservative, emulsifying wax,glycerin, isopropyl palmitate, lactic acid, purified water, and sorbitolsolution. In addition, the compositions may contain polyethylene glycol400. They may be mixed to form ointments with, for example, benzylalcohol 2% (wt/wt) as preservative, white petrolatum, emulsifying wax,and tenox II (butylated hydroxyanisole, propyl gallate, citric acid,propylene glycol). Woven pads or rolls of bandaging material, e.g.,gauze, may be impregnated with the compositions in solution, lotion,cream, ointment, or other such form may also be used for topicalapplication. The compositions may also be applied topically using atransdermal system, such as one of an acrylic-based polymer adhesivewith a resinous crosslinking agent impregnated with the composition andlaminated to an impermeable backing.

Examples of suitable skin contact adhesive materials include, but arenot limited to, polyethylenes, polysiloxanes, polyisobutylenes,polyacrylates, polyurethanes, and the like. Alternatively, thedrug-containing reservoir and skin contact adhesive are separate anddistinct layers, with the adhesive underlying the reservoir that, inthis case, may be either a polymeric matrix as described above, or be aliquid or hydrogel reservoir, or take some other form.

Additional Administration Forms. Additional dosage forms of thisinvention include dosage forms as described in U.S. Pat. Nos. 6,340,475;6,488,962; 6,451,808; 5,972,389; and 5,007,790. Additional dosage formsof this invention also include dosage forms as described in U.S. PatentApplication Nos. 20030147952, 20030104062, 20030104053, 20030044466,20030039688, and 20020051820. Additional dosage forms of this inventionalso include dosage forms as described in PCT Application Nos. WO03/35041, WO 03/35040, WO 03/35029, WO 03/35177, WO 03/35039, WO02/96404, WO 02/32416, WO 01/97783, WO 01/56544, WO 01/32217, WO98/55107, WO 98/11879, WO 97/47285, WO 93/18755, and WO such formsincorporated by reference.

Solutions: After an H₂S donor has been selected, it may be dissolvedinto a solution. The solution may be an aqueous-based solution, such aswater, saline, or the like. In some variations, other fluids andsolutions may be appropriate.

Various formulations of saline are known in the art and may be used withthe present invention. For example, the saline may be lactated Ringer'ssolution, acetated Ringer's solution, phosphate buffered saline (PBS),Dulbecco's phosphate buffered saline (D-PBS), Tris-buffered saline(TBS), Hank's balanced salt solution (HBSS), or Standard saline citrate(SSC).

The saline solutions of the present invention are, in certainembodiments, “normal saline” (i.e., a solution of about 0.9% w/v ofNaCl). Normal saline has a slightly higher degree of osmolality comparedto blood; however, in various embodiments, the saline may be isotonic inthe body of a subject such as a human patient. In certain embodiments,“half-normal saline” (i.e., about NaCl) or “quarter-normal saline”(i.e., about 0.22% NaCl) may be used with the present invention.Optionally, about 5% dextrose or about 4.5 g/dL of glucose may beincluded in the saline. In various embodiments, one or more salt,buffer, amino acid and/or antimicrobial agent may be included in thesaline.

In various embodiments, a preservative or stabilizer may be included inthe composition or solution. For example, the prevention of the actionof microorganisms may be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (for example, methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, EDTA, metabisulfite, benzyl alcohol, thimerosal, orcombinations thereof. Agents that may be included suitable for useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile solutions or dispersions (U.S.Pat. No. 5,466,468, specifically incorporated herein by reference in itsentirety). In all cases the composition is preferably sterile and mustbe fluid to facilitate easy injectability. Solutions are preferablystable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. Examples of stabilizers which may be includedinclude buffers, amino acids such as glycine and lysine, carbohydratessuch as dextrose, mannose, galactose, fructose, lactose, sucrose,maltose, sorbitol, mannitol, and the like. Appropriate stabilizers orpreservatives may be selected according to the route of administrationdesired. A particle filter or microbe filter may be used and may benecessary according to the route of administration desired.

The weight ranges of compounds in the solution may vary. For example, invarious embodiments, the composition may comprise about 0.1-10 wt %,more preferably 1-5 wt % H₂S donor, about 1-5 wt %preservative/stabilizer, about 1-5 wt % NaCl, and about 85%-97% water.The ratio of H₂S donor to water may be varied as needed to achieve thedesired treatment of the endothelial dysfunction condition.

The solution and/or composition may also be sterilized prior toadministration. Methods for sterilization are well known in the art andinclude heating, boiling, pressurizing, filtering, exposure to asanitizing chemical (for example, chlorination followed bydechlorination or removal of chlorine from solution), aeration,autoclaving, and the like.

The H₂S donor may be formulated into a solution in any number of ways.For example, it may be solubilized by agitation or by sonication, orother methods known in the art. After the H₂S donor has beensolubilized, it may be administered to a subject in need of treatment ofan endothelial dysfunction condition. In certain embodiments, an H₂Sdonor is admixed with a solution in a closed vacuum container, and thecombined solutions are then mechanically agitated for 3-5 minutes andheld in a thermo-neutral sonicator until use.

In certain embodiments, solutions of the present invention may be acomponent of an emulsion, such as a water-in-oil or an oil-in-wateremulsion, including a lipid emulsion, such as a soybean oil emulsion.Certain emulsions have been described previously for intravenous (daSilva Telles, et al., 2004, Rev. Bras. Anaestesiol Campianas 54(5):2004)or epidural administration (Chai et al. 2008, British J Anesthesia100:109-115), such described emulsion techniques incorporated byreference herein.

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more H₂S donors dissolved or dispersed in apharmaceutically acceptable carrier. The phrases “pharmaceutical orpharmacologically acceptable” refers to molecular entities andcompositions that do not produce an adverse, allergic, or other untowardreaction when administered to an animal, such as, for example, a human,as appropriate. The preparation of a pharmaceutical composition thatcontains at least one H₂S donor in solution or additional activeingredient will be known to those of skill in the art in light of thepresent disclosure, as exemplified by “Remington: The Science andPractice of Pharmacy,” 20th Edition (2000), which is incorporated hereinby reference in its entirety. Moreover, for animal (for example, human)administration, it will be understood that preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biological Standards.

In various embodiments, the compositions of the present inventionfurther comprise cyclodextrin. Cyclodextrins are a general class ofmolecules composed of glucose units connected to form a series ofoligosaccharide rings (See Challa et al., 2005, AAPS PharmSciTech6:E329-E357). In nature, the enzymatic digestion of starch bycyclodextrin glycosyltransferase (CGTase) produces a mixture ofcyclodextrins comprised of 6, 7 and 8 anhydroglucose units in the ringstructure (α-, β-, and γ-cyclodextrin, respectively). Commercially,cyclodextrins are also produced from starch, but different, morespecific enzymes are used. Cyclodextrins have been employed informulations to facilitate the delivery of cisapride, chloramphenicol,dexamethasone, dextromethorphan, diphenhydramine, hydrocortisone,itraconazole, and nitroglycerin (Welliver and McDonough, 2007, Sci WorldJ, 7:364-371). In various embodiments, the cyclodextrin of the inventionis hydroxypropyl-Beta-cyclodextrin, sulfobutylether-beta-cyclodextrin,alpha-dextrin or combinations thereof. In certain embodiments,cyclodextrin may be used as a solubilizing agent.

In various other embodiments, compositions of the present invention maycomprise human serum albumin purified from plasma, or recombinant humanserum albumin. In certain embodiments, human serum albumin may be usedas a solubilizing agent. In other embodiments, the compositions of theinvention may comprise propylene glycol. In other embodiments, thecompositions of the invention may comprise perfluorooctyl bromide. Inother embodiments, the compositions of the invention may compriseperfluorocarbon. In certain embodiments, perfluorocarbon may be used asa solubilizing agent.

In various embodiments, a preservative or stabilizer may be included inthe composition or solution. For example, the prevention of the actionof microorganisms may be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (for example, methylparabens, propylparabens), chlorobutanol,phenol, sorbic acid, EDTA, metabisulfite, benzyl alcohol, thimerosal, orcombinations thereof. Agents which may be included suitable for useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile solutions or dispersions (U.S.Pat. No. 5,466,468, specifically incorporated herein by reference in itsentirety). In all cases the composition is preferably sterile and mustbe fluid to facilitate easy injectability. Solutions are preferablystable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. Examples of stabilizers which may be includedinclude buffers, amino acids such as glycine and lysine, carbohydratessuch as dextrose, mannose, galactose, fructose, lactose, sucrose,maltose, sorbitol, mannitol, etc. Appropriate stabilizers orpreservatives may be selected according to the route of administrationdesired. A particle filter or microbe filter may be used and may benecessary according to the route of administration desired.

Administration of the disclosed compositions in a method of treatmentmay be achieved in a number of different ways, using methods known inthe art. Such methods include, but are not limited to, topicallyadministering solutions, suspensions, creams, pastes, oils, lotions,gels, foam, hydrogel, ointment, liposomes, emulsions, liquid crystalemulsions, and nano-emulsions.

The therapeutic and prophylactic methods of the invention thus encompassthe use of pharmaceutical compositions of the invention. Theformulations of the pharmaceutical compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient into association with a carrier or one ormore other accessory ingredients, and then, if necessary or desirable,shaping or packaging the product into a desired single- or multi-doseunit. For example, unit dose container may be such that an H₂S donorsolution is contained in a crushable sealed ampoule which in turn isenclosed in protective covering on which pressure is applied to crushthe ampoule which then releases the H₂S donor solution for percolationthrough a flint-type tip which capped the ampoule in protectivecovering. When such packaging configuration is employed, care is takento leave as little as possible or ideally no headspace in ampoule forany volatile portion of the solution to escape and cause a change insolution composition over a period of shelf life.

Although the description of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts, including mammals. Modificationof pharmaceutical compositions suitable for administration to humans inorder to render the compositions suitable for administration to variousanimals is well understood, and the ordinarily skilled veterinarypharmacologist may design and perform such modification with merelyordinary, if any, experimentation. Subjects to which administration ofthe pharmaceutical compositions of the invention is contemplatedinclude, but are not limited to, humans and other primates, mammalsincluding commercially relevant mammals such as non-human primates,cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor ophthalmic, vaginal, topical, intranasal, buccal, or another routeof administration.

A pharmaceutical composition of the invention may be prepared, packaged,or sold in bulk, as a single unit dose, or as a plurality of single unitdoses. A unit dose is discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject or a convenientfraction of such a dosage such as, for example, one-half or one-third ofsuch a dosage.

The relative amounts of the active ingredient, the pharmaceuticallyacceptable carrier, and any additional ingredients in a pharmaceuticalcomposition of the invention will vary, depending upon the identity,size, and condition of the subject treated and further depending uponthe route by which the composition is to be administered. By way ofexample, the composition may comprise between 0.1% and 100% (w/w) activeingredient.

In addition to the active ingredient, a pharmaceutical composition ofthe invention may further comprise one or more additionalpharmaceutically active agents. Non-limiting examples of such anadditional pharmaceutically active agents are fluorouracil cream,imiquimod cream, ingenol mebutate gel, diclofenac sodium gel, topicalretinoids, and tirbanibulin (Klisyri) ointment.

Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

Formulations of a pharmaceutical composition suitable for topicaladministration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Formulations may be prepared, packaged, or sold in unitdosage form, such as in ampules, crushable or otherwise, or inmulti-dose containers containing a preservative. Formulations fortopical administration include, but are not limited to, suspensions,solutions, emulsions in oily or aqueous vehicles, solutions,suspensions, creams, pastes, oils, lotions, gels, foam, hydrogel,ointment, liposomes, emulsions, liquid crystal emulsions, nanoemulsions,implantable sustained-release or biodegradable formulations. Suchformulations may further comprise one or more additional ingredientsincluding, but not limited to, suspending, stabilizing, or dispersingagents.

The pharmaceutical compositions may be prepared, packaged, or sold inthe form of a sterile aqueous or oily suspension or solution. Thissuspension or solution may be formulated according to the known art, andmay comprise, in addition to the active ingredient, additionalingredients such as the dispersing agents, wetting agents, or suspendingagents described herein. Such sterile formulations may be prepared usinga non-toxic acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, butare not limited to, Ringer's solution, isotonic sodium chloridesolution, and fixed oils such as synthetic mono- or di-glycerides. Otherformulations that are useful include those which comprise the activeingredient in a liposomal preparation, or as a component of abiodegradable polymer system. Compositions for sustained release orimplantation may comprise pharmaceutically acceptable polymeric orhydrophobic materials such as an emulsion, an ion exchange resin, asparingly soluble polymer, or a sparingly soluble salt.

In some embodiments, the pharmaceutical compositions of the inventionmay be contained in a crushable ampule irrespective of the route ofdelivery to the patient.

It is contemplated that any embodiment discussed in this specificationmay be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions of the inventionmay be used to achieve methods of the invention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific procedures, embodiments, claims, and examples described herein.Such equivalents were considered to be within the scope of thisinvention and covered by the claims appended hereto. For example, itshould be understood, that modifications in reaction conditions,including but not limited to reaction times, reaction size/volume, andexperimental reagents, such as solvents, catalysts, pressures,atmospheric conditions, e.g., nitrogen atmosphere, andreducing/oxidizing agents, with art-recognized alternatives and using nomore than routine experimentation, are within the scope of the presentapplication.

Dosing Regimes:

The present methods for treating endothelial dysfunctions are carriedout by administering a therapeutic for a time and in an amountsufficient to result in decreased endothelial dysfunction.

The amount and frequency of administration of the compositions can varydepending on, for example, what is being administered, the state of thepatient, and the manner of administration. In therapeutic applications,compositions can be administered to a patient suffering from endothelialdysfunction in an amount sufficient to relieve or least partiallyrelieve the symptoms of the endothelial dysfunction and itscomplications. The dosage is likely to depend on such variables as thetype and extent of progression of the endothelial dysfunction, theseverity of the endothelial dysfunction, the age, weight and generalcondition of the particular patient, the relative biological efficacy ofthe composition selected, formulation of the excipient, the route ofadministration, and the judgment of the attending clinician. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test system. An effective dose is a dose thatproduces a desirable clinical outcome by, for example, improving a signor symptom of the endothelial dysfunction or slowing its progression.

The amount of therapeutic per dose can vary. For example, a subject canreceive from about 0.1 μg/kg to about 10,000 μg/kg. Generally, thetherapeutic is administered in an amount such that the peak plasmaconcentration ranges from 150 nM-250 μM. Exemplary dosage amounts canfall between 0.1-5000 μg/kg, 100-1500 μg/kg, 100-350 μg/kg, 340-750μg/kg, or 750-1000 μg/kg. Exemplary dosages can 0.25, 0.5, 0.75, 1°, or2 mg/kg. In another embodiment, the administered dosage can range from0.05-5 mmol of therapeutic (e.g., 0.089-3.9 mmol) or 0.1-50 μmol oftherapeutic (e.g., 0.1-25 μmol or 0.4-20 μmol).

The plasma concentration of therapeutic can also be measured accordingto methods known in the art. Exemplary peak plasma concentrations oftherapeutic can range from 0.05-10 μM, 0.1-10 μM, 0.1-5.0 μM, or 0.1-1μM. Alternatively, the average plasma levels of therapeutic can rangefrom 400-1200 μM (e.g., between 500-1000 μM) or between 50-250 μM (e.g.,between μM). In some embodiments where sustained release of the drug isdesirable, the peak plasma concentrations (e.g., of therapeutic) may bemaintained for 6-14 hours, e.g., for 6-12 or 6-10 hours. In otherembodiments where immediate release of the drug is desirable, the peakplasma concentration (e.g., of therapeutic) may be maintained for, e.g.,30 minutes.

The frequency of treatment may also vary. The subject can be treated oneor more times per day with therapeutic (e.g., once, twice, three, fouror more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12,or 24 hours). Preferably, the pharmaceutical composition is administered1 or 2 times per 24 hours. The time course of treatment may be ofvarying duration, e.g., for two, three, four, five, six, seven, eight,nine, ten or more days. For example, the treatment can be twice a dayfor three days, twice a day for seven days, twice a day for ten days.Treatment cycles can be repeated at intervals, for example weekly,bimonthly or monthly, which are separated by periods in which notreatment is given. The treatment can be a single treatment or can lastas long as the life span of the subject (e.g., many years).

Kits: Any of the pharmaceutical compositions of the invention describedherein can be used together with a set of instructions, i.e., to form akit. The kit may include instructions for use of the pharmaceuticalcompositions as a therapy as described herein. For example, theinstructions may provide dosing and therapeutic regimes for use of thecompounds of the invention to reduce symptoms and/or underlying cause of

The invention illustratively disclosed herein suitably may explicitly bepracticed in the absence of any element which is not specificallydisclosed herein. While various embodiments of the present inventionhave been described in detail, it is apparent that various modificationsand alterations of those embodiments will occur to and be readilyapparent those skilled in the art. However, it is to be expresslyunderstood that such modifications and alterations are within the scopeand spirit of the present invention, as set forth in the appendedclaims. Further, the invention(s) described herein is capable of otherembodiments and of being practiced or of being carried out in variousother related ways. The present disclosure also contemplates otherembodiments “comprising,” “consisting of” and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not. In addition, it is to be understood that thephraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including,” “comprising,” or “having” and variations thereof herein ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items, while only the terms “consisting of” and“consisting only of” are to be construed in the limitative sense.

Wherefore, I/we claim:
 1. A method of treating or preventingmethamphetamine-related endothelial dysfunction in a patient, comprisingadministering to the patient an effective dose of a pharmacologiccomposition comprising: a hydrogen sulfide (H₂S) donor, or a salt,solvate, ester, amide, clathrate, stereoisomer, enantiomer, prodrug oranalog thereof; and cystathionine gamma lyase (CSE), or a salt, solvate,ester, amide, clathrate, stereoisomer, enantiomer, prodrug, analog, orsynthetic mRNA coded to increase CSE expression.
 2. The method of claim1 wherein the H₂S donor is selected from the group consisting of sodiumsulfide, diallyl trisulfide, diallyl disulfide, acillin, sugammadex,sulfanilamide, disulfram, sulfonamide, sulfinates, sulfoxides,persulfides, polysulfides, and sulfones.
 3. The method of claim 1,wherein the therapeutic is administered in one of oral, intravenous, andtransdermal pathways.
 4. The method of claim 1, wherein the H₂S donor isadministered in a dosage of between 0.5 mg/kg and 10.0 mg/kg.
 5. Themethod of claim 1, wherein the H₂S donor is administered exactly once ina dosage period, the dosage period being between 1 day and 30 days. 6.The method of claim 1, wherein the therapeutic has an enteric coating.7. The method of claim 1, wherein the H₂S donor is formulated for one oforal and peritoneal administration and is administered in a dosage ofbetween 1.0 mg and 100.0 mg of H₂S donor.
 8. A method of treating orpreventing methamphetamine-related endothelial dysfunction in a patientcomprising: administering to the patient an effective dose of apharmacologic composition comprising a therapeutic, the therapeuticincluding a hydrogen sulfide (H₂S) donor, or a salt, solvate, ester,amide, clathrate, stereoisomer, enantiomer, prodrug or analog thereof,wherein the H₂S donor is selected from the group consisting of sodiumsulfide, diallyl trisulfide, diallyl disulfide, acillin, sulfanilamide,disulfram, sulfonamide, sulfinates, sulfoxides, persulfides,polysulfides, and sulfones.
 9. The method of claim 8, wherein thetherapeutic is administered in one of oral, intravenous, and transdermalpathways.
 10. The method of claim 8, wherein the H₂S donor isadministered in a dosage of between 0.5 mg/kg and 10.0 mg/kg.
 11. Themethod of claim 8, wherein the H₂S donor is administered exactly once ina dosage period, the dosage period being between 1 day and 30 days. 12.The method of claim 8, wherein the therapeutic has an enteric coating.13. The method of claim 8, wherein the H₂S donor is formulated for oneof oral and peritoneal administration and is administered in a dosage ofbetween 1.0 mg and 100.0 mg of H₂S donor.
 14. A method of treating anendothelial dysfunction-related disease in a methamphetamine patientcomprising: administering to the patient an effective dose of apharmacologic composition comprising a therapeutic, the therapeuticincluding a hydrogen sulfide (H₂S) donor, or a salt, solvate, ester,amide, clathrate, stereoisomer, enantiomer, prodrug or analogs thereof;wherein the endothelial dysfunction-related disease is selected from thegroup consisting of atherosclerosis, hypertension, myocardialinfarction, diabetes, and cardiovascular disease, or a preconditionthereof.
 15. The method of claim 14, wherein the therapeutic furthercomprises cystathionine gamma lyase (CSE), or a salt, solvate, ester,amide, clathrate, stereoisomer, enantiomer, prodrug, analog, orsynthetic mRNA coded to increase CSE expression.
 16. The method of claim14, wherein the therapeutic is administered in one of oral, intravenous,and transdermal pathways.